Biofilm-removing antimicrobial compositions and uses thereof

ABSTRACT

The present invention provides novel compositions comprising: (a) a metaperiodate and (b) chlorhexidine or a chlorohexidine salt and uses thereof for the preparation of devices, and in particular medical devices, susceptible to colonization by biofilm forming bacteria.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.13/145,991 filed Sep. 8, 2011, which is a national phase ofInternational Patent Application No. PCT/CA2010/000067 filed Jan. 22,2010, which claims priority from U.S. Application No. 61/146,852 filedJan. 23, 2009, all of which are incorporated by reference herein intheir entirety.

FIELD OF THE INVENTION

The present invention relates to novel antimicrobial compositions thatinhibit growth and proliferation of biofilm embedded microorganisms, andmethods/uses thereof.

BACKGROUND

Biofilms are medically and industrially important because they canaccumulate on a wide variety of substrates and are resistant toantimicrobial agents and detergents. Microbial biofilms develop whenmicroorganisms adhere to a surface and produce extracellular polymersthat facilitate adhesion and provide a structural matrix. Thereforeinhibiting adhesion to surfaces is important. This surface may be inert,non-living material or living tissue.

A method of long-term prevention from biofilm formation is needed. Alsoneeded is a composition that allows for low quantities of a compositionto be used effectively to reduce toxicity or other side effects to auser or patient. There is also a need for compositions that aremedically acceptable, effective at lower concentrations, free ofresistance and relatively economical to manufacture on a commercialscale for reducing biofilm formation in biomedical devices.

SUMMARY OF THE INVENTION

An embodiment of the invention includes a composition for inhibitingmicrobial biofilms comprising: (a) a cationic peptide and (b) aquaternary ammonium compound, a silver containing particle, or5-fluorouracil.

A cationic peptide includes omiganan, protamine, Cecropin A, or acombination thereof. A cationic peptide can be low molecular weightderivatives or salts of protamine such as protamine sulfate. Acomposition comprising protamine sulfate can include comprise about 100μg/ml to about 1000 μg/ml of protamine sulfate. In an embodiment, thecomposition can be concentrated and then diluted prior to use.

Quaternary ammonium compounds can be derivatives or salts ofbenzalkonium such as benzalkonium chloride. The amount of benzalkoniumchloride included in the composition can be about 20 μg/ml to about 200μg/ml. In an embodiment, the composition can be concentrated and thendiluted prior to use.

The amount of 5-fluoruracil included in the composition can compriseabout 50 μg/ml to about 5000 μg/ml of 5-fluoruracil. In an embodiment,the composition can be concentrated and then diluted prior to use.

Silver containing particles includes a silver nanoparticle or silversulfadiazine. The amount of silver included in the composition cancomprise about 1 μg/ml to about 1000 μg/ml of silver. In an embodiment,the composition can be concentrated and then diluted prior to use.

An embodiment of the invention includes a composition for inhibitingmicrobial biofilms comprising: (a) a metaperiodate and (b)5-fluorouracil, silver containing particle, chlorhexidine, or triclosan.

The amount of chlorhexidine included in the composition can compriseabout 1 μg/ml to about 100 μg/ml of chlorhexidine. In an embodiment, thecomposition can be concentrated and then diluted prior to use.

Metaperiodate can be sodium or potassium meta-periodate. The amount ofsodium metaperiodate included in the composition can comprise about 20μg/ml to about 2000 μg/ml of meta-periodate. In an embodiment, thecomposition can be concentrated and then diluted prior to use.

The concentration of chlorhexidine or chlorohexidine salt may be about10 μg/ml to about 10000 μg/ml chlorhexidine or chorhexidine salt, about100 μg/ml to about 5000 μg/ml chlorhexidine or chorhexidine salt, orabout 1000 μg/ml to about 5000 μg/ml chlorhexidine or chorhexidine salt,about 100 μg/ml to about 1000 μg/ml chlorhexidine or chorhexidine salt,or about 500 μg/ml chlorhexidine or chorhexidine salt.

A composition according to the invention can be effective againstbiofilms produced by microbial species including S. epidermidis, E.faecalis, E. coli, P. mirabilis, P. aeruginosa, K. pneumoniae, S.aureus, S. viridans, K. oxytoca, S. saprophyticus, Legionellapneumophila, Mycobacterium spp., Citrobacter freundii, Aeromonashydrophile, Fusobacterium nucleatum, Vancomycin resistant Enterococcusfaecalis (VRE), Enterococcus faecium, Actinomyces naeslundii,Enterobacter cloacae, Providencia stuartii, Serratia marcescens, orcombinations thereof. In a further embodiment of the invention, thecomposition is effective against biofilms produced by gram-negativebacterial species.

In another embodiment of the invention, a composition can be effectiveagainst biofilms produced by gram-positive bacterial species.

In yet another embodiment of the invention, a composition can beeffective against biofilms produced by fungal species, including C.albicans.

In another embodiment, a method comprises administering a composition ofthe invention to disinfect an article of matter, such as dentalinstruments, medical instruments, medical devices, surfaces (e.g.,tabletop, countertop, bathtub, tile, etc.), tubing, and the like. Acomposition of the invention can comprise a compound of the inventionand common household disinfectants.

A method of the invention includes rinsing, coating a surface, orimpregnating a surface of an object with at least one surface. An objectcan include a medical device including disposable or permanent orindwelling catheters, long term urinary devices, tissue bonding urinarydevices, wound drain tubes, ventricular catheters, endotracheal tubes,breathing tubes, feeding tubes, dairy lines, and drinking water lines.Furthermore, a method of the invention includes cleaning pipelines inindustries such as food and beverage industries, paper mills, coolingtowers, and gas and oil industries.

In yet another embodiment, a method comprises treating wounds byadministering a composition of the invention, wherein the woundsinclude, but are not limited to, cutaneous abscess, surgical wounds,sutured lacerations, contaminated lacerations, burn wounds such aspartial and full thickness burns, decubitus ulcers, stasis ulcers, legulcers, foot ulcers, venous ulcers, diabetic ulcers, ischemic ulcers,and pressure ulcers.

One embodiment of the present invention includes a method comprisingcoating treating, or impregnating composition in wound care devices suchas non-resorbable gauze/sponge dressing, hydrophilic wound dressing,occlusive wound dressing, hydrogel wound, and burn dressing. The presentinvention also includes use of a spray-applicator containing acomposition of the invention as a wound care device.

A method of the invention also includes treating a patient with amicrobial infection by administering a composition of the invention,wherein the composition is coated or impregnating on the surface of anobject. An object can be a wound care device such as non-resorbablegauze/sponge dressing, hydrophilic wound dressing, occlusive wounddressing, hydrogel wound, and burn dressing. The present invention alsoincludes use of a spray-applicator containing a composition of theinvention as a wound care device.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph illustrating the effect of control (solutionwithout an active ingredient), 12 μg/ml of protamine sulfate (PS), 6μg/ml of benzalkonium chloride (BZK) and a combination of 12 μg/mlprotamine sulfate and 6 μg/ml benzalkonium chloride (PS+BZK) on growthand biofilm formation of Escherichia coli.

FIG. 2 is a bar graph illustrating the effect of control (solutionwithout an active ingredient), 25 μg/ml of protamine sulfate (PS), 12.5μg/ml of benzalkonium chloride (BZK) and a combination of 25 μg/mlprotamine sulfate and 12.5 μg/ml benzalkonium chloride (PS+BZK) ongrowth and biofilm formation of Pseudomonas aeruginosa.

FIG. 3 is a bar graph illustrating the effect of control (solutionwithout an active ingredient), 1.5 μg/ml of protamine sulfate (PS), 0.8μg/ml of benzalkonium chloride (BZK) and a combination of 1.5 μg/mlprotamine sulfate and 0.8 μg/ml benzalkonium chloride (PS+BZK) on growthand biofilm formation of Staphylococcus epidermidis.

FIG. 4 is a bar graph illustrating the effect of control (solutionwithout an active ingredient), 250 μg/ml of sodium metaperiodate (SMP),6.25 μg/ml of 5-fluorouracil (FU) and a combination of 250 μg/ml sodiummetaperiodate and 6.25 μg/ml 5-fluorouracil (SMP+FU) on growth andbiofilm formation of Escherichia coli.

FIG. 5 is a bar graph illustrating the effect of control (solutionwithout an active ingredient), 250 μg/ml of sodium metaperiodate (SMP),3.75 μg/ml of 5-fluorouracil (FU) and a combination of 250 μg/ml sodiummetaperiodate and 3.75 μg/ml 5-fluorouracil (SMP+FU) on growth andbiofilm formation of Pseudomonas aeruginosa.

FIG. 6 is a bar graph illustrating the effect of control (solutionwithout an active ingredient), 250 μg/ml of sodium metaperiodate (SMP),20 μg/ml of 5-fluorouracil (FU) and a combination of 250 μg/ml sodiummetaperiodate and 20 μg/ml 5-fluorouracil (SMP+FU) on growth and biofilmformation of Staphylococcus epidermidis.

FIG. 7 is a bar graph illustrating the effect of control (solutionwithout an active ingredient), 250 μg/ml of sodium metaperiodate (SMP),1 μg/ml of chlorhexidine (CHX) and a combination of 250 μg/ml sodiummetaperiodate and 1 μg/ml chlorhexidine (SMP+CHX) on growth and biofilmformation of Escherichia coli.

FIG. 8 is a bar graph illustrating the effect of control (solutionwithout an active ingredient), 125 μg/ml of sodium metaperiodate (SMP),6 μg/ml of chlorhexidine (CHX) and a combination of 125 μg/ml sodiummetaperiodate and 6 μg/ml chlorhexidine (SMP+CHX) on growth and biofilmformation of Pseudomonas aeruginosa.

FIG. 9 is a bar graph illustrating the effect of control (solutionwithout an active ingredient), 250 μg/ml of sodium metaperiodate (SMP),0.5 μg/ml of chlorhexidine (CHX) and a combination of 250 μg/ml sodiummetaperiodate and 0.5 μg/ml chlorhexidine (SMP+CHX) on growth andbiofilm formation of Staphylococcus epidermidis.

DETAILED DESCRIPTION OF THE INVENTION

Antimicrobial compositions have found an increasing number of commercialand consumer uses. An effective antimicrobial composition, such as acomposition that inhibits growth and proliferation of biofilm embeddedmicroorganisms, is useful in a plethora of applications. Such anantimicrobial composition can either be used on its own, incorporatedinto a consumable, or incorporated into a surface desirable to be freeof bacteria.

Definitions

The term “antimicrobial” refers to a compound or a composition thatkills or slows/stops the growth of microorganisms, including, but notlimited to bacteria and yeasts.

The term “biofilm embedded microorganisms” refers to any microorganismthat forms a biofilm during colonization and proliferation on a surface,including, but not limited to, gram-positive bacteria (e.g., S.epidermidis), gram-negative bacteria (e.g., P. aeruginosa), and/or fungi(e.g., C. albicans).

The term “biofilm formation” refers to the attachment of microorganismsto surfaces and the subsequent development of multiple layers of cells.

The term “inhibition” or “inhibiting” refers to a decrease of biofilmassociated microorganism formation and/or growth. The microorganisms caninclude bacteria (e.g., streptococci) or fungi (e.g., Candida spp.)

“Modulating detachment” refers to increasing or decreasing bacterial orfungal biofilm detachment or release of bacterial or fungal cells from abiofilm. Further, “modulating detachment”, is also inclusive of changesin the ability of the bacteria or fungal to attach as a biofilm.

The term “mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic, farm, sport and zooanimals, or pet animals, such as dogs, horses, cats, cattle, pigs,sheep, etc.

The term “therapeutically effective amount” refers to an amount of acomposition of the invention effective to “alleviate” or “treat” adisease or disorder in a subject or mammal. A “therapeutically effectiveamount” as used herein also includes an amount that is bacteriostatic orbacteriocidal, for example, an amount effective for inhibiting growth ofbiofilm associated bacteria or killing biofilm associated bacteria,respectively.

A “therapeutically effective amount” as used herein also includes anamount that is fungistatic or fungicidal, for example, an amounteffective for inhibiting further growth of biofilm associated fungi orkilling biofilm associated fungi, respectively. By administering thecomposition suitable for use in methods of the invention concurrentlywith an antimicrobial compound, the therapeutic antimicrobial compoundmay be administered in a dosage amount that is less than the dosageamount required when the therapeutic antimicrobial compound isadministered as a sole active ingredient. By administering lower dosageamounts of the active ingredient, the side effects associated therewithshould accordingly be reduced.

A “prophylactically effective amount” as used herein includes an amounteffective for preventing or protecting against infectious diseases, andsymptoms thereof, and amounts effective for alleviating or treatinginfectious diseases, related diseases, and symptoms thereof.

The term “treatment”, “treating”, or “alleviating” refers to anintervention performed with the intention of altering or inhibiting thepathology of a disorder.

The term “preventing” or “prophylaxis” refers to preventing disease,pathology, and/or symptoms.

The term “dental caries” refers to a localized destruction of tissues ofa tooth by acid produced from bacterial degradation of fermentablesugars. The chief etiological agent of dental caries is S. mutans.Degradation of fermentable sugars by S. mutans on the tooth surfaceproduces an acid that destroys oral tissues, and more particularly,enamel and dentin.

The term “dental plaque” is a general term for the diverse microbialcommunity (predominantly bacteria) found on the tooth surface, embeddedin a matrix of polymers of bacterial and salivary origin. Further,“dental plaque-associated S. mutans” refers to S. mutans that is acomponent of the dental plaque.

The term “endocarditis” refers to an infection of the endocardialsurface of the heart, which may include one or more heart valves, themural endocardium, or a septal defect.

The term “gingivitis” refers to inflammation of gingival tissue withoutloss of connective tissue.

The term “oral diseases” refers to diseases and disorders affecting theoral cavity or associated medical conditions. Oral diseases include, butare not limited to, dental caries; periodontal diseases (e.g.,gingivitis, adult periodontitis, early-onset periodontitis, etc.);mucosal infections (e.g., oral candidiasis, herpes simplex virusinfections, recurrent aphthous ulcers, etc.); oral and pharyngealcancers; and precancerous lesions.

The term “periodontal disease” refers to an inflammatory process of thegingival tissues and/or periodontal membrane of the teeth, resulting ina deep gingival sulcus, possibly producing periodontal pockets and lossof alveolar bone.

The term “periodontitis” refers to inflammation and loss of connectivetissue of the supporting or surrounding structure of teeth with loss ofattachment.

The term “chronic wound” refers to a wound that fails to progressthrough an orderly and timely sequence of repair or a wound that doesnot respond to treatment and/or the demands of treatment are beyond thepatient's physical health, tolerance or stamina. Many wounds that arefirst considered to be acute wounds ultimately become chronic wounds dueto factors still not well understood. One significant factor is thetransition of planktonic bacteria within the wound to form a biofilm.

In the context of wound treatment, “biofilm disruption” or “inhibitionof biofilm reconstitution” refers to biofilm clearance from a chronic oracute wound, or to inhibit reconstitution of a biofilm mass fromremnants remaining after debridement and thereby promote healing of awound.

Compositions

An embodiment of the invention includes a composition for inhibiting ordisrupting microbial biofilms comprising: (a) a cationic peptide and (b)a quaternary ammonium compound, a silver containing particle orantineoplastic agents. A cationic peptide includes omiganan, protamine,Cecropin A, or a combination thereof. A cationic peptide can also be alow molecular weight derivative or salt of protamine such as protaminesulfate.

A quaternary ammonium compound includes, but is not limited to,benzalkonium chloride, benzalkonium bromide, benzalkonium saccharinate,cetalkonium chloride, cetealkonium bromide, hydrogenated tallowalkoniumchloride, tallowalkonium chloride, didecyldimethylammonium saccharinate,hex adecylpyridinium saccharinate, benzalkonium acesulfamate,didecyldimethylammonium acesulfamate, hexadecylpyridinium acesulfamate,3-hydroxy-1-octyloxymethylpyridinium acesulfamate,3-hydroxy-1-octyloxymethylpyridinium saccharinate, cetylpyridiniumchloride, or a combination thereof. In an embodiment of the invention, aquaternary ammonium compound comprises benzalkonium chloride.

A silver containing particle can be silver nanoparticles or silversulfadiazine.

An embodiment of the invention also includes a composition forinhibiting or dispersing microbial biofilms comprising: (a) anantibiofilm compound and (b) antineoplastic agent, silver containingparticle, bis-guanide, or triclosan. An antibiofilm compound includesprotamine sulfate, metaperiodate, N-acetylecysteine,ethylenediaminetetraacetic acid (EDTA), ovotransferrin, lactoferrin,proteolytic and polysaccharide degrading enzymes, RNAIII inhibitorypeptide (RIP), or gallium. In an embodiment of the invention, anantibiofilm compound comprises sodium or potassium metaperiodate.

An antineoplastic agent can be mitomycin c, 5-fluorouracil, bleomycin,doxorubicin, or a combination thereof. In a preferred embodiment, theantineoplastic agent is comprises 5-fluorouracil.

A bis-guanide compound can be chlorhexidine (its base or salts),alexidine, or polyhexamethylene biguanides.

A composition comprising benzalkonium chloride includes, for example,between 20 μg/ml to 200 μg/ml of benzalkonium chloride. The higher endof this stated range can be used to prepare a concentrated product thatwould be diluted prior to use.

The amount of 5-fluoruracil included in the composition can compriseabout 50 μg/ml to about 5000 μg/ml of 5-fluoruracil. In an embodiment,the composition can be concentrated and then diluted prior to use.

The amount of chlorhexidine included in the composition can compriseabout 1 μg/ml to about 100 μg/ml of chlorhexidine. In an embodiment, thecomposition can be concentrated and then diluted prior to use.

Silver containing particles can include a silver nanoparticle or silversulfadiazine. The amount of silver included in the composition cancomprise about 1 μg/ml to about 1000 μg/ml of silver. In an embodiment,the composition can be concentrated and then diluted prior to use.

Metaperiodate can be sodium or potassium meta-periodate. The amount ofsodium metaperiodate included in the composition can comprise about 20μg/ml to about 2000 μg/ml of meta-periodate. The higher end of thisstated range can be used to prepare a concentrated product that would bediluted prior to use.

In an embodiment of the invention, a composition according to theinvention is effective against biofilms produced by microbial speciessuch as S. epidermidis, E. faecalis, E. coli, P. mirabilis, P.aeruginosa, K. pneumoniae, S. aureus, S. viridans, K. oxytoca, S.saprophyticus, L. pneumophila, Mycobacterium spp., C. freundii, A.hydrophile, F. nucleatum, A. naeslundii, P. stuartii, S. marcescens, orcombinations thereof. In a further embodiment of the invention, acomposition is effective against biofilms produced by gram-negativebacterial species.

In another embodiment of the invention, a composition is effectiveagainst biofilms produced by gram-positive bacterial species.

In yet another embodiment of the invention, a composition is effectiveagainst biofilms produced by fungal species, including C. albicans.

Compositions described herein have enhanced antibiofilm activity whencombined. Enhanced antibiofilm activity is evidenced by the smallquantities of each of these compounds that can be used to produce aneffective antimicrobial composition, less than would be required if anyof the compounds were to be used on their own. Low levels and increasedefficacy of the active compounds or ingredients make this invention verydesirable and relatively economical to manufacture. Thus, typicalbacterial resistances for antibiotics may not develop.

Higher concentrations of these compounds can be used if it is desiredfor certain applications and will depend on the bacteria targeted andthe device to be treated. Lower concentrations of compounds may also beused in certain situations.

While the active components discussed herein may be 100% of thecomposition of the invention, a composition can contain from at leastabout 1% to about 50% of active components by weight based upon totalweight of the composition of the invention being employed. In oneembodiment, a composition comprises from at least about 0.5% to about25% (by weight) active components.

Compositions of the invention may include any number of well knownactive components and base materials. Compositions may further compriseingredients such as, but not limited to: suitable solvents such aswater, and ethanol; antimicrobials such antibacterials and antifungals;binding, bonding, coupling agent, or cross-linking agent; or a pHadjuster.

Other possible components of the composition include, but are notlimited to buffer solutions, phosphate buffered saline, saline, water,polyvinyl chloride, polyethylene, polyurethane, polypropylene, silicone(e.g., silicone elastomers and silicone adhesives), polycarboxylic adds,(e.g., polyacrylic add, polymethacrylic add, polymaleic add,poly-(maleic acid monoester), polyaspartic acid, polyglutamic acid,aginic acid or pectimic acid), polycarboxylic acid anhydrides (e.g.,polymaleic anhydride, polymethacrylic anhydride or polyacrylic acidanhydride), polyamines, polyamine ions (e.g., polyethylene imine,polyvinylamine, polylysine, poly-(dialkylamineoethyl methacrylate),poly-(dialkylaminomethyl styrene) or poly-(vinylpyridine)), polyammoniumions (e.g., poly-(2 methacryloxyethyl trialkyl ammonium ion),poly-(vinylbenzyl trialkyl ammonium ions), poly-(N.N.-alkylypyridiniumion) or poly-(dialkyloctamethylene ammonium ion) and polysulfonates(e.g. poly-(vinyl sulfonate) or poly-(styrene sulfonate)), collodion,nylon, rubber, plastic, polyesters, polyethylene terephthalateoptionally sealed with gelatin, collagen, or albumin,polytetrafluoroethylene, latex, and derivatives thereof, elastomers,cyanoacrylates, methacrylates, papers with porous barrier films,adhesives, e.g., hot melt adhesives, solvent based adhesives, andadhesive hydrogels, fabrics, and crosslinked and non-crosslinkedhydrogels, and any other polymeric materials which facilitate dispersionof the active components and adhesion of the biofilm penetrating coatingto at least one surface of the medical device. Linear copolymers,cross-linked copolymers, graft polymers, and block polymers, containingmonomers as constituents of the above-exemplified polymers may also beused.

In another embodiment, the composition can further comprise one or moredisinfecting agents. Disinfects can comprise alcohols (such as ethanolor isopropanol), aldehydes (such as glutaraldehyde), oxidizing agents(such as sodium hypochlorite, calcium hypochlorite, chloramine, hydrogenperoxide, iodine, peracetic acid, performic acid, potassiumpermanganate, and potassium peroxymonosulfate), phenolics (such asphenol, o-phenylphenol, chloroxylenol, hexachlorophene, and thymol). Adisinfectant can be a spray or a liquid. A disinfectant can beconcentrated or ready-to-use. A disinfectant can be for commercial orhousehold use. A composition of the invention can also be incorporatedinto household disinfectants, laundry detergent, and household cleaningsolutions.

Biofilm on Surfaces

Biofilms accumulate on the surface of various objects such as medicaldevices, tubing, pipelines, counter/tabletops, filters, water lines, andtiles of various kinds.

Biofilms on indwelling medical devices may be composed of gram-positiveor gram-negative bacteria or yeasts. Bacteria commonly isolated fromthese devices include (a) gram-positive Enterococcus faecalis,Vancomycin resistant Enterococcus faecalis, Staphylococcus epidermidis,Staphylococcus aureus, and Enterococcus faecium; (b) gram-negativeEscherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobactercloacae, and Pseudomonas aeruginosa; and (c) fungal species, such asCandida albicans. The organisms most commonly isolated from urinarycatheter biofilms are S. epidermidis, E. faecalis, E. coli, mirabilis,P. aeruginosa and K. pneumoniae. In the case of vascular catheters, S.aureus and S. epidermidis account for almost 70-80% of all infectiousorganisms, with S. epidermidis being the most common organism. C.albicans accounts for about 10-15% of catheter infections. Gram-negativebacilli account for almost 60-70%, Enterococci for about 25%, and C.albicans for about 10% of cases of urinary tract infections.Catheter-associated urinary tract infection is a very common nosocomialinfection (about 1 million patients in US hospitals each year). It isthe second most common cause of nosocomial infections (Maki and Tambyah,Emerg. Infect. Dis., 7:1-6, 2001).

A composition of present invention can be useful for decontaminating,inhibiting growth, or preventing growth on surfaces where microorganismsform a biofilm, such as tubing. A method of the invention includesrinsing or decontaminating a surface by contacting the surface with acomposition of the invention. Further, a method of the inventionincludes inhibiting biofilm growth or preventing biofilm growth byincorporating a composition of the invention into a surface. Acomposition of the invention can be incorporated into a surface bycoating or impregnating the surface of the object.

A composition of the invention can coat, impregnate, flush, or rinse asurface of tubing or a medical device, especially an insertable medicaldevice, Tubing includes, but is not limited to, disposable, permanent,and indwelling catheters, long term urinary devices, tissue bondingurinary devices, wound drain tubes, ventricular catheters, endotrachealtubes, breathing tubes, feeding tubes, dairy lines, oil and gas pipelineand drinking water lines. Some tubing can also be considered a medicaldevice. Insertable medical devices include catheters, which can beinspected without invasive techniques such as endoscopy. Medical devicesmay be formed of any suitable metallic materials or non-metallicmaterials known to persons skilled in the art. Examples of metallicmaterials include, but are not limited to, tivanium, titanium, andstainless steel, and derivatives or combinations thereof, Examples ofnon-metallic materials include, but are not limited to, thermoplastic orpolymeric materials such as rubber, plastic, polyesters, polypropylene,polycarbonate, polyvinyl chloride, nylon, polyethylene, polyurethane,silicone, polyethylene terephthalate, polytetrafluoroethylene, latex,elastomers and polyethylene terephthalate sealed with gelatin, collagenor albumin, and derivatives or combinations thereof. Medical devicesinclude at least one surface for applying a composition of theinvention. Preferably, a composition of the invention is applied to anentire medical device. Compositions can also be incorporated intopolymers, which are used to form devices such as catheters byimpregnating or by drug polymer conjugation.

Furthermore, a composition of the invention can also be used to cleanpipelines in industries such as food and beverage industries, papermills, cooling towers and gas and oil industries by contacting a surfacewith biofilm growth.

A composition of the invention can also be incorporated into vacuumsystems and vacuum filters, paint and wall coverings, humidifiers andhumidifier filters, and vacuum cleaners, toys incorporation intoplastics for a variety of household items, including the inside andoutside of washing machines, dishwashers, animal water dishes, bathroomtiles and fixtures, sealants and grout, towels, home and kitchencontainers, dishes, cutting boards, dish drying trays, bathtubsincluding aerated bathtubs and hot tub spas, fish ponds, swimming pools,bird baths, garden hose, planters and hot tubs.

Industrial applications to antimicrobial compounds include their use indairy lines, either as a flush or wash for such lines, or incorporatedwithin the lines, for example as a coating; liquid distribution lines inthe food and beverage manufacturing or dispensing, for example, use as acoating in feeder lines for high sugar or syrup distribution in themanufacturing of soft drinks; pulp and paper mills (for biofouling); inthe manufacturing and containment of cosmetics from production lineequipment down to the end consumable, either incorporated within thecosmetic or coated on the jar containing the cosmetic; in watertreatment facilities; in the leaching process used in mining; to preventcorrosion caused or accelerated by organisms, in oil and gas pipelines,in the souring of oil fields, and in cooling towers.

Consumer and light commercial uses of antimicrobial agents include theirincorporation in general household disinfectants; laundry detergent;cleaning supplies; equipment involved in the leeching process or mining;wound care; a vacuum system; HVAC systems; vacuum cleaner bags; paintcovering; wall coverings; window frames; doors; door frames; coolingtowers; humidifiers; vacuum cleaners; filters such as a vacuum filter, ahumidifier filter, hot tub filter, or a swimming pool filter; toys;cosmetic containers; plastic bottles; water jugs; a tap and water spout;incorporation into plastics for a variety of household items includingthe inside and outside of washing machines and dishwashers; animal waterdishes; bathroom tiles and fixtures; sinks; showers; shower heads;toilets; toilets lids; toilet seats; sealants and grout; towels; homeand kitchen containers; dishes; cups; utensils such as forks, spoons,knives, and spatulas; bowls; food storage containers; beverage storagecontainers; cutting boards; dish drying trays; garbage bags; bathtubsincluding whirlpool bathtubs and hot tub spas; sinks; showers; fishponds; swimming pools; swimming pool liners; swimming pool skimmer; pondliners; bird baths; garden hose; water sprinkling lines; planters; andhot tubs. In another aspect, the present invention provides acomposition suitable for coating an object which is desirable to bemicroorganism resistant, for example paint, wall covering, or protectiveplastic coating. The object may be any object which is desirable to bemicroorganism resistant, such as a home product, an industrial product,a medical product or medical device, a piece of apparel or a textile, abuilding product, etc.

An embodiment includes, a method to reduce, inhibit, or prevent amicrobial biofilm comprising disinfecting, cleaning, or rinsing asurface by contacting said surface with a combination of a compositionof the invention and a disinfectant. A combination of a composition ofthe invention and a disinfectant can also include a hydrophilic polymer.Preferably, the hydrophilic polymer is a nitrogen-containing polymerhaving surface-modifying properties. The method can also reduce,inhibit, or prevent deposits on a surface. Deposits can be lime scale,soap scum and other organically encrusted or flocculated deposits. Asurface can be a hard surface, preferably silicone surface, morepreferably a glass or ceramic surface, or metal surface.

A method of incorporating a composition of the invention into a surfaceof an object includes immersing or flushing, coating, drug-polymerconjugate and impregnating (Tunny et al., Rev. Med. Microbiol.,74:195-205, 1996). In a clinical setting, suitable catheters can betreated by immersion immediately prior to placement, which offersflexibility and control to clinicians in certain situations.

Direct incorporation of a composition of the invention into a polymericmatrix of a medical device at the polymer synthesis stage or at thedevice manufacture stage is also possible (Schierholz et al.,Biomaterials, 18:839-844, 1997).

In a preferred embodiment, an object, such as a medical device, issubmerged in a composition for at least 5 minutes. Alternatively, anobject may be flushed with a composition. When an object is tubing(e.g., dental unit waterline, a dairy line, a food and beverageprocessing line, etc.), a composition of the invention may be pouredinto the tubing and both ends of the tubing clamped such that thecomposition is retained within the lumen of the tubing. The tubing isthen allowed to remain filled with the composition for a period of timesufficient to remove substantially all of the microorganisms from atleast one surface of the object, generally, for at least about 1 minuteto about 48 hours. Alternatively, tubing may be flushed by pouring acomposition of the invention into the lumen of the tubing for an amountof time sufficient to prevent substantial growth of all biofilm embeddedmicroorganisms. Such flushing may be required only once, or may berequired at regular intervals over the lifetime of use of the tubing.Concentrations of active components in a composition may vary as desiredor necessary to decrease the amount of time the composition is incontact with a medical device.

In another aspect, a method of the invention includes coating a medicaldevice. Broadly, coating a medical device includes applying acomposition coating to at least one surface of the medical device in anamount sufficient to substantially reduce growth, proliferation, orcolonization of biofilm microorganisms on at least one surface of themedical device.

In one specific embodiment, at least one surface of a medical device iscontacted with a composition of the invention under conditions whereinthe composition of the invention covers at least one surface of themedical device. “Contacting” includes, but is not limited to,impregnating, compounding, mixing, integrating, coating, spraying anddipping.

In another embodiment of coating a medical device, combining activeingredients and a base material at room temperature and mixing acomposition of the invention for a time sufficient to evenly disperseactive agents in the composition prior to applying the composition to asurface of the device form the composition coating. A medical device maybe contacted with a composition of the invention for a period of timesufficient for the composition to adhere to at least one surface of thedevice. After a composition of the invention is applied to a surface ofthe device, it is allowed to dry.

Although one layer or coating of the composition is believed to providethe desired composition coating, multiple layers or coatings can beapplied. Multiple layers of a composition of the invention can beapplied to the at least one surface of a medical device by repeating thesteps discussed above. For example, a medical device is contacted with acomposition of the invention three times, allowing the composition todry on at least one surface of the medical device prior to contactingthe medical device with the composition for each subsequent layer. Inother words, the medical device can include three coats, or layers, ofthe composition on at least one surface of the medical device. Further,coatings or layers are also possible.

In another embodiment, a method for coating a medical device with acomposition of the invention includes incorporating the composition intothe material forming the medical device during formation of the medicaldevice. For example, the composition may be combined with materialsforming the medical device, e.g., silicone, polyurethane, polyvinylchloride, polyethylene, polytetrafluoroethylene, polyethyleneterephthalate, and/or polypropylene, and extruded with the materialforming the medical device, thereby incorporating the composition intomaterial forming the medical device. In this embodiment, the compositionmay be incorporated in a septum or adhesive, which is placed at amedical device insertion or implantation site.

Examples of devices that can be coated using the compositions of theinvention include tubing and other surface medical devices, such asurinary catheter, mucous extraction catheter, suction catheter,umbilical cannula, contact lenses, intrauterine devices, intravaginaland intraintestinal devices, endotracheal tubes, bronchoscopes, dentalprostheses and orthodontic devices, surgical instruments, dentalinstruments, tubing, dental water lines, dental drain tubes, fabrics,paper, indicator strips (e.g., paper indicator strips or plasticindicator strips), adhesives (e.g., hydrogel adhesives, hot-meltadhesives, or solvent-based adhesives), bandages, tissue dressings orhealing devices and occlusive patches, and any other surface devicesused in the medical field. Devices may include electrodes, externalprostheses, fixation tapes, compression bandages, and monitors ofvarious types. Medical devices also include any device that may beplaced at the insertion or implantation site such as the skin near theinsertion or implantation site, and which include at least one surfacewhich is susceptible to colonization by biofilm embedded microorganisms.In one specific embodiment, a composition of the invention is integratedinto an adhesive, such as tape, thereby providing an adhesive, which mayprevent growth or proliferation of biofilm embedded microorganisms on atleast one surface of the adhesive. Medical devices for the presentinvention include surfaces of equipment in operating rooms, emergencyrooms, hospital rooms, clinics, and bathrooms.

In another aspect, the invention provides a method for reducing biofilmmicroorganisms from at least one surface of the medical device. In onespecific embodiment, the method of reducing biofilm formation from atleast one surface of the medical device includes contacting the medicaldevice with a composition of the invention. “Contacting” includes, butis not limited to, soaking, rinsing, flushing, submerging, and washing.A medical device should be contacted with a composition of the inventionfor a period of time sufficient to substantially reduce a biofilm fromat least one surface of a medical device. In one specific embodiment, amedical device is submerged in a composition for at least 5 minutes.Alternatively, a medical device may be flushed with a composition. Inthe example of tubing, such as dental drain tubing (dental water line),a composition of the invention may be poured into the dental draintubing and both ends of the tubing clamped such that the composition isretained within the lumen of the tubing. The tubing is then allowed toremain filled with the composition for a period of time sufficient tosubstantially reduce or remove all of the microorganisms from at leastone surface of the medical device, generally, for at least about 1minute to about 48 hours. Alternatively, the tubing may be flushed bypouring the composition into the lumen of the tubing for an amount oftime sufficient to substantially reduce or remove all biofilm growth.

The concentration of active ingredients in a composition of theinvention may vary as desired or necessary to decrease the amount oftime the composition is in contact with the medical device.

In embodiments of the invention, a step of forming a composition of theinvention may also include one or more of steps of adding an organicsolvent, a medical device material penetrating agent, or an alkalinizingagent to the composition in order to enhance reactivity of a surface ofa medical device with the composition. In an embodiment of coatingmedical devices, an organic solvent, medical device material penetratingagent, and/or alkalinizing agent preferably facilitate adhesion of acomposition of the invention to at least one surface of a medicaldevice.

In one embodiment, a device is placed in contact with a composition ofthe invention by dipping the device in the composition for about 30minutes to about 120 minutes at a temperature from about 35° C. to about65° C. A device may be placed in contact with a composition of theinvention by dipping the device in the composition for about 120 minutesat a temperature of about 45° C. The device is then removed from thecomposition, and the composition on the surface of the device is allowedto dry. The medical device may be placed in an oven or other heatedenvironment for a period of time sufficient for the composition to dry.

In another embodiment, a method for coating medical devices with acomposition of the invention includes forming a coating of an effectiveconcentration to substantially reduce the growth, proliferation, orcolonization of biofilm microorganisms on at least one surface of themedical device by dissolving an active ingredient in an organic solvent,combining a medical device material penetrating agent to activeingredients and organic solvent, and combining an alkalinizing agent toimprove reactivity of a surface of the medical device. The compositionis then heated to a temperature of about 35° C. to about 65° C. toenhance adherence of the composition to at least one surface of thedevice. A composition coating is applied to at least one surface of themedical device, for example, contacting a coating of a composition ofthe invention to the at least one surface of a medical device for asufficient period of time for the composition coating to adhere to atleast one surface of the medical device. The medical device is removedfrom the composition coating and allowed to dry for at least 8 hours,and preferably overnight, at room temperature. The medical device maythen be rinsed with a liquid, such as water, and allowed to dry for atleast 2 hours, and preferably 8 hours, before being sterilized. Tofacilitate drying of a composition onto a surface of a medical device,the medical device may be placed into a heated environment such as anoven.

Wound Care

Wounds often have multiple barriers to healing. Wound healing andinfection is influenced by the relationship between the ability ofbacteria to create a stable, prosperous community within a woundenvironment and the ability of the host to control the bacterialcommunity. Since bacteria are rapidly able to form their own protectivemicroenvironment (biofilm) following their attachment to a surface, theability of the host to control these organisms is likely to decrease asthe biofilm community matures. Within a stable biofilm community,interactions between aerobic and anaerobic bacteria are likely toincrease their net pathogenic effect, enhancing their potential to causeinfection and delay healing. Biofilms have been linked to chronicwounds: microscopic evaluation of chronic wounds showed well organizedbiofilm with extracellular polymeric substance adhered around colonybacteria in at least 60% of the chronic wounds (Mertz, Wounds, 15: 1-9,2003).

In addition to a direct effect on wound healing by the production ofdestructive enzymes and toxins, mixed communities of microorganisms mayalso indirectly affect healing by promoting a chronic inflammatorystate. Prolonged exposure to bacteria within a chronic wound leads to aprolonged inflammatory response, resulting in the release of freeradicals and numerous lytic enzymes that could have a detrimental effecton cellular processes involved in wound healing. Proteinases releasedfrom a number of bacteria, particularly P. aeruginosa, are known toaffect growth factors and many other tissue proteins that are necessaryfor the wound healing process (Steed et al., J. Am. Coll. Surg,183:61-64, 1996; Travis et al., Trends Microbiol. 3:405-407, 1995). Theincreased production of exudates that often accompanies increasedmicrobial load has been associated with the degradation of growthfactors and matrix metalloproteinases (MMPs), which subsequently affectcell proliferation and wound healing (Falanga et al., J Invest Dermatol.1:125-127, 1994).

A method of the invention includes treating, cleaning, or disinfecting awound by administering a composition of the invention. A method of theinvention also includes treating, cleaning, or disinfecting a wound bycontacting the wound with a wound care device of the invention, whereinthe wound care device includes a composition of the invention. Suchwounds include chronic wounds, acute wounds, surgical wounds, surgicalsites, second and third degree burns, stasis ulcers, tropic lesions,decubitus ulcers, severe cuts, and abrasions. Wound care devicesinclude, but are not limited to, dressings and bandages. A compositionof the invention can also be formulated as a gel or an ointment.

Caries/Periodontal Diseases

Caries and periodontal diseases are two of the most common chronicinfectious diseases affecting humankind and are associated with dentalplaque, which is a biofilm on tooth surfaces. Streptococci account forapproximately 20% of the salivary bacteria, which include Streptococcusspp. such as Streptococcus mutans, Streptococcus sobrinus, Streptococcussanguis, Streptococcus gordonii, Streptococcus oralis and Streptococcusmitis. Although four streptococci, S. mutans, S. sobrinus, S. sanguisand S. oralis are directly involved in the initiation of dental caries,S. mutans is considered to be the principal etiological agent of caries(Devulapalle et al., Carbohydr. Res., 339:1029-1034, 2004). Periodontaldisease comprises a collection of inflammatory conditions of theperiodontium (gingiva, periodontal ligament, cementum, and alveolarbone) due to a chronic bacterial infection, i.e., dental plaque. Over90% of the population of the United States is affected by periodontaldisease (Brown et al., J. Dent. Res. 75:672-683, 1996).

In an embodiment, a composition of the invention can be incorporatedinto an oral consumable product such as toothpaste, mouth wash, chewinggum, breath mints, dental floss, dentures, mouth guards and similarconsumables.

In an embodiment, a method includes treating or preventing caries orperiodontal disease in a subject comprising administering a compositionof the invention to the subject. In a further embodiment, a methodincludes treating or preventing caries or periodontal disease in asubject by contacting the biofilm with an oral consumable productcomprising a composition of the invention. Periodontal disease includesgingivitis, periodontitis, and acute necrotizing ulcerative gingivitis.

Although the invention has been described with reference to illustrativeembodiments, it is to be understood that the invention is not limited tothese precise embodiments, and that various changes and modification areto be intended to be encompassed in the appended claims.

EXAMPLES Example 1 Effect of Protamine Sulfate (PS) and BenzalkoniumChloride (BZK) Alone and in Combination on Escherichia coli Growth andBiofilm Formation

An overnight broth culture of E. coli was grown in TSB and used asinoculum. 96-well microplates containing colony forming antigen medium(for gram-positive species) in the absence and the presence of eachcompound (PS or BZK) separately and together (PS and BZK) wereinoculated and incubated at 26° C. for 24 hours. Growth of planktoniccells based on absorbance at 600 nm using Labsystems Multiskan Ascentmicroplate reader was determined. Biofilm was measured by discarding themedia in the wells, rinsing the well three times with water, andstaining the bound cells with crystal violet. The dye was thensolubilized with 33% acetic acid, and absorbance at 630 nm wasdetermined using a microtiter plate reader. A composition comprising PSand BZK showed an enhanced inhibitory effect on biofilm formation, ascompared to either PS or BZK alone (FIG. 1; Table 1).

TABLE 1 Inhibitory effect of protamine sulfate (PS; 12 μg/ml) andbenzalkonium chloride (BZK; 6 μg/ml) alone and in combination onEscherichia coli biofilm* Expected Biofilm Unexpected Biofilm BiofilmInhibition by Inhibition by Inhibition by (Additive) (More thanadditive) Pathogen PS BZK PS + BZK PS + BZK E. coli 0.04 0.14 0.19 0.32*As determined by the reduction in biofilm formation in terms of OpticalDensity (OD) at 630 nm.

Example 2 Effect of Protamine Sulfate (PS) and Benzalkonium Chloride(BZK) Alone and in Combination on Pseudomonas aeruginosa Growth andBiofilm Formation

An overnight broth culture of P. aeruginosa was grown in TSB and used asinoculum. 96-well microplates containing colony forming antigen medium(for gram-positive species) in the absence and the presence of eachcompound (PS or BZK) separately and together (PS and BZK) wereinoculated and incubated at 26° for 24 hours. Growth of planktonic cellsbased on absorbance at 600 nm using Labsystems Multiskan Ascentmicroplate reader was determined. Biofilm was measured by discarding themedia in the wells, rinsing the well three times with water, andstaining the bound cells with crystal violet. The dye was thensolubilized with 33% acetic acid, and absorbance at 630 nm wasdetermined using a microtiter plate reader. A composition comprising PSand BZK showed an enhanced inhibitory effect on biofilm formation, ascompared to either PS or BZK alone (FIG. 2; Table 2).

TABLE 2 Inhibitory effect of protamine sulfate (PS; 25 μg/ml) andbenzalkonium chloride (BZK; 12.5 μg/ml) alone and in combination onPseudomonas aeruginosa biofilm* Expected Biofilm Unexpected BiofilmBiofilm Inhibition by Inhibition by Inhibition by (Additive) (Morethanadditive) Pathogen PS BZK PS + BZK PS + BZK P. aeruginosa 0.00 0.000.00 0.18 *As determined by the reduction in biofilm formation in termsof Optical Density (OD) at 630 nm.

Example 3 Effect of Protamine Sulfate (PS) and Benzalkonium Chloride(BZK) Alone and in Combination on Staphylococcus epidermidis Growth andBiofilm Formation

An overnight broth culture of S. epidermidis was grown in TSB, and usedas inoculum. 96-well microplates containing TSB (for gram-positivespecies) in the absence and the presence of each compound (PS or BZK)separately and together (PS and BZK) were inoculated and incubated at37° C. for 24 hours. Growth of planktonic cells based on absorbance at600 nm using Labsystems Multiskan Ascent microplate reader wasdetermined. Biofilm was measured by discarding the media in the wells,rinsing the well three times with water, and staining the bound cellswith crystal violet. The dye was then solubilized with 33% acetic acid,and absorbance at 630 nm was determined using a microtiter plate reader.A composition comprising PS and BZK showed an enhanced inhibitory effecton biofilm formation, as compared to either PS or BZK alone (FIG. 3;Table 3).

TABLE 3 Inhibitory effect of protamine sulfate (PS; 1.5 μg/ml) andbenzalkonium chloride (BZK; 0.8 μg/ml) alone and in combination onStaphylococcus epidermidis biofilm* Expected Biofilm Unexpected BiofilmBiofilm Inhibition by Inhibition by Inhibition by (Additive) (More thanadditive) Pathogen PS BZK PS + BZK PS + BZK S. epidermidis 0.34 1.211.54 1.90 *As determined by the reduction in biofilm formation in termsof Optical Density (OD) at 630 nm.

Example 4 Effect of Sodium Metaperiodate (SMP) and 5-Fluorouracil (FU)Alone and in Combination on Escherichia coli Growth and BiofilmFormation

An overnight broth culture of E. coli was grown in TSB and used asinoculum. 96-well microtiter plate containing TSB in the absence andpresence of each compound separately (SMP or FU) and together (SMP+FU)were inoculated. The biofilm was grown by incubating at 37° C. for 24hours. Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. Acomposition comprising SMP and FU showed an enhanced inhibitory effecton E. coli biofilm formation, as compared to either SMP or FU alone(FIG. 4; Table 4).

TABLE 4 Inhibitory effect of sodium metaperiodate (SMP; 250 μg/ml) and5-fluorouracil (FU; 6.25 μg/ml) alone and in combination on Escherichiacoli biofilm* Expected Biofilm Unexpected Biofilm Biofilm Inhibition byInhibition by Inhibition by (Additive) (More than additive) Pathogen SMPFU SMP + FU SMP + FU E. coli 0.3 0.17 0.21 0.25 *As determined by thereduction in biofilm formation in terms of Optical Density (OD) at 630nm

Example 5 Effect of Sodium Metaperiodate (SMP) and 5-Fluorouracil (FU)Alone and in Combination on Pseudomonas aeruginosa Growth and BiofilmFormation

An overnight broth culture of P. aeruginosa was grown in TSB and used asinoculum. 96-well microtiter plate containing TSB in the absence andpresence of each compound separately (SMP or FU) and together (SMP+FU)were inoculated. The biofilm was grown by incubating at 37° C. for 24hours. Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. Acomposition comprising SMP and FU showed an enhanced inhibitory effecton P. aeruginosa biofilm formation, as compared to either SMP or FUalone (FIG. 5; Table 5).

TABLE 5 Inhibitory effect of sodium metaperiodate (SMP; 250 μg/ml) and5-fluorouracil (FU; 3.75 μg/ml) alone and in combination on Pseudomonasaeruginosa biofilm* Expected Biofilm Unexpected Biofilm BiofilmInhibition by Inhibition by Inhibition by (Additive) (More thanadditive) Pathogen SMP FU SMP + FU SMP + FU P. aeruginosa 0.16 0.00 0.162.37 *As determined by the reduction in biofilm formation in terms ofOptical Density (OD) at 630 nm.

Example 6 Effect of Sodium Metaperiodate (SMP) and 5-Fluorouracil (FU)Alone and in Combination on Staphylococcus epidermidis Growth andBiofilm Formation

An overnight broth culture of S. epidermidis was grown in TSB and usedas inoculum. 96-well microtiter plate containing TSB in the absence andpresence of each compound separately (SMP or FU) and together (SMP+FU)were inoculated. The biofilm was grown by incubating at 37° C. for 24hours. Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. Acomposition comprising SMP and FU showed an enhanced inhibitory effecton S. epidermidis biofilm formation, as compared to either SMP or FUalone (FIG. 6; Table 6).

TABLE 6 Inhibitory effect of sodium metaperiodate (SMP; 250 μg/ml) and5-fluorouracil (FU; 20 μg/ml) alone and in combination on Staphylococcusepidermidis biofilm* Expected Biofilm Unexpected Biofilm BiofilmInhibition by Inhibition by Inhibition by (Additive) (More thanadditive) Pathogen SMP FU SMP + FU SMP + FU S. epidermidis 0.00 1.431.43 2.22 * As determined by the reduction in biofilm formation in termsof Optical Density (OD) at 630 nm.

Example 7 Effect of Sodium Metaperiodate (SMP) and Chlorhexidine (CHX)Alone and in Combination on Escherichia coli Growth and BiofilmFormation

An overnight broth culture of E. coli was grown in TSB and used asinoculum. 96-well microtiter plate containing TSB in the absence andpresence of each compound separately (SMP or CHX) and together (SMP+CHX)were inoculated. The biofilm was grown by incubating at 37° C. for 24hours. Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. Acomposition comprising SMP and CHX showed an enhanced inhibitory effecton E. coli biofilm formation, as compared to either SMP or CHX alone(FIG. 7; Table 7).

TABLE 7 Inhibitory effect of sodium metaperiodate (SMP; 250 μg/ml) andchlorhexidine (CHX; 1 μg/ml) alone and in combination on Escherichiacoli biofilm* Expected Biofilm Unexpected Biofilm Biofilm Inhibition byInhibition by Inhibition by (Additive) (More than additive) Pathogen SMPCHX SMP + CHX SMP + CHX E. coli 0.12 0.04 0.16 0.22 *As determined bythe reduction in biofilm formation in terms of Optical Density (OD) at630 nm.

Example 8 Effect of Sodium Metaperiodate (SMP) and Chlorhexidine (CHX)Alone and in Combination on Pseudomonas aeruginosa Growth and BiofilmFormation

An overnight broth culture of bacteria and yeast were grown in TSB andused as inoculum. 96-well microplates containing TSB (for gram-positivespecies and yeast) and colony forming unit antigen medium (forgram-negative bacteria) in the absence and the presence of each compoundseparately (SMP or CHX) and together (SMP+CHX) were inoculated. Thebiofilm was grown by incubating at 37° C. (for gram-positive bacteriaand yeast) and 26° C. (for gram-negative bacteria) for 24 hours. Growthof planktonic cells based on absorbance at 600 nm using LabsystemsMultiskan Ascent microplate reader was determined. Biofilm was measuredby discarding the media in the wells, rinsing the well three times withwater, and staining the bound cells with crystal violet. The dye wasthen solubilized with 33% acetic acid, and absorbance at 630 nm wasdetermined using a microtiter plate reader. A composition comprising SMPand CHX showed an enhanced inhibitory effect on P. aeruginosa biofilmformation, as compared to either SMP or CHX alone (FIG. 8; Table 8).

TABLE 8 Inhibitory effect of sodium metaperiodate (SMP; 125 μg/ml) andchlorhexidine (CHX; 6 μg/ml) alone and in combination on Pseudomonasaeruginosa biofilm* Expected Biofilm Unexpected Biofilm BiofilmInhibition by Inhibition by Inhibition by (Additive) (More thanadditive) Pathogen SMP CHX SMP + CHX SMP + CHX P. aeruginosa 0.17 0.660.83 0.88 *As determined by the reduction in biofilm formation in termsof Optical Density (OD) at 630 nm

Example 9 Effect of Sodium Metaperiodate (SMP) and Chlorhexidine (CHX)Alone and in Combination on Staphylococcus epidermidis Growth andBiofilm Formation

An overnight broth culture of S. epidermidis was grown in TSB and usedas inoculum. 96-well microtiter plate containing TSB in the absence andpresence of each compound separately (SMP or CHX) and together (SMP+CHX)were inoculated. The biofilm was grown by incubating at 37° C. for 24hours. Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. Acomposition comprising SMP and CHX showed an enhanced inhibitory effecton S. epidermidis biofilm formation, as compared to either SMP or CHXalone (FIG. 9; Table 9).

TABLE 9 Inhibitory effect of sodium metaperiodate (SMP; 250 μg/ml) andchlorhexidine (CHX; 0.5 μg/ml) alone and in combination onStaphylococcus epidermidis biofilm* Expected Biofilm Unexpected BiofilmBiofilm Inhibition by Inhibition by Inhibition by (Additive) (More thanadditive) Pathogen SMP CHX SMP + CHX SMP + CHX S. epidermidis 0.00 0.000.00 0.24 *As determined by the reduction in biofilm formation in termsof Optical Density (OD) at 630 nm

Example 10 Antimicrobial Activity of Protamine Sulfate (PS) and SilverNanoparticles (SNP) Alone and in Combination Against MedicalDevice-Associated Pathogens

Bacterial strains were grown overnight at 37° C. with 100 rpm shaking inTryptic Soy Broth (TSB) and diluted to approximately 10⁵ CFU/ml. Assayswere conducted by methods of minimum inhibitory concentration (MIC) in96-well microtiter plates as described previously (Amsterdam, D. 1996,In: V. Loman, Ed., “Antibiotics in laboratory medicine”, p. 52-111,Williams and Wilkins, Baltimore, M.D.). Antimicrobials PS and SNP bothalone and together were serially diluted in TSB (100 μl), and 100 μl ofbacterial suspension was added to each well. Plates were incubated at37° C. for 24 hours and read at 600 nm using a microtiter plate reader(Multiskan Ascent, Labsystems, Helsinki, Finland). A compositionscomprising PS and SNP showed an enhanced inhibitory effect on bacterialgrowth as compared to either PS or SNP above, as shown in Table 10.

TABLE 10 Minimal inhibitory concentrations (MICs) of protamine sulfate(PS), silver nanoparticles (SNP) alone and in combination againstmedical device associated pathogens MIC (μg/ml) Pathogen PS SNP PS + SNPS. aureus >200 >1000 100 + 500 Escherichia coli >200 >1000  200 + 1000

Example 11 Broad-Spectrum Antimicrobial Activity of Sodium Metaperiodate(SMP) and Chlorhexidine (CHX) Disinfectant Formulation

A disinfectant was formulated containing 0.15% chlorhexidine (CHX) and0.025% sodium metaperiodate (SMP). The antimicrobial activity was testedagainst Gram positive and Gram negative bacteria using the AOAC “usedilution” method. Stainless steel penicylinders were soaked for 15minutes in a broth culture of the test organism to form a bacterialfilm. The carriers with bacterial film were then removed and dried forapproximately 30 minutes at 37° C. This now represents a nonporous,hard, inanimate surface contaminated with a dried film of bacteria. Thecontaminated carriers were exposed to 10 ml of the disinfectant for 10minutes at 20° C. After treatment; the carriers were removed from thedisinfectant and placed in 10 ml subculture broth media containingappropriate neutralizers. The subculture tubes were incubated 48 hoursat 37° C. The tubes were examined for growth as determined by theturbidity of the media. The disinfectant formulation was effectiveagainst all the pathogens tested.

TABLE 11 Broad-Spectrum Antimicrobial Activity of SMP-CHX DisinfectantFormulation on Gram Positive and Gram Negative Bacteria Number ofCarriers Test Organism Exposed Showing Growth Staphylococcus aureus(ATCC 6538) 180 0 Pseudomonas aeruginosa (ATCC 15442) 180 0 Salmonellacholeraesuis (ATCC 10708) 180 0 Escherichia coli O157:H7  20 0 E. coli(ATCC 25404)  20 0 Acinetobacter baumannii  20 0 Methicillin-resistantS. aureus (MRSA)  20 0 Vancomycin-resistant Enterococci (VRE)  20 0Listeria monocytogenes  20 0 Klebsiella pneumoniae  20 0 Campylobacterjejuni (ATCC 33560)  20 0

We claim:
 1. A composition for removing or reducing a microbial biofilmformed by S. epidermidis, the composition comprising: (a) ametaperiodate or a metaperiodate salt in a concentration of about 250μg/ml and (b) chlorhexidine or a chlorohexidine salt in a concentrationof about 0.5 μg/ml.
 2. The composition of claim 1, wherein themetaperiodate is preferably selected from sodium metaperiodate orpotassium metaperiodate.
 3. The composition according to claim 1,wherein said composition is incorporated in a product selected from thegroup consisting of a toothbrush; dental floss; a denture, a mouthguard; a dairy line; a dairy line filter; a water line; a line used infood and beverage manufacturing; a general household disinfectant; alaundry detergent; cleaning supplies; equipment involved in the leechingprocess or mining; wound care; a vacuum system; an HVAC system; a vacuumcleaner bag; paint covering; a wall covering; a window frame; a door; adoor frame; a cooling tower; a humidifier; a vacuum cleaning a filter; atoy; a cosmetic container; a plastic bottle; a water jug; a tap andwater spout; a washing machine; a dishwasher; an animal water dish; abathroom tile; a bathroom fixture; a sink; a shower; a shower heat; atoilet; a toilet lid; a toilet seat; a sealant; grout; a towel; a homeand kitchen container; a dish; a cup; an utensil; a bowl; a food storagecontainer; a beverage storage container; a cutting board; a dish dryingtray; a garbage bag; a bathtub; a whirlpool; an aerated bathtub; a sink;a shower; a fish pond; a swimming pool; a swimming pool liner; aswimming pool skimmer; a pond liner; a bird bath; a garden hose; a watersprinkling line; a planter; and a hot tub.
 4. The composition accordingto claim 1, further comprising an additive selected from the groupconsisting of water, ethanol, bleach constituent or an oxidizingconstituents, a binding or bonding or coupling agent, a wetting agent,an odor absorbing agent, a levelling agent, an adherent, a thickener, anantistatic agent, an optical brightening compound, an opacifier, anucleating agent, an antioxidant, a UV stabilizer, a filler, a permanentpress finish, a softener, a lubricant, a curing accelerator, anadhesive, a bis-phenol, a biguanide, a silver compound, a5-fluorouracil, a bisphosphonate, a gallium compound, a bismuthiol, achelating agent, a cationic polypeptide, a quaternary ammonium compound,a maleimide, an antibiotic, a pH adjuster, a buffer solution, phosphatebuffered saline, saline, polyvinyl chloride, polyethylene, polyurethane,polypropylene, silicone, a polycarboxylic acid, a polycarboxylic acidanhydride, a polyamine, a polyamine ions, a polyammonium ion, andpolysulfonates, collodion, nylon, rubber, plastic, a polyester,polyethylene tetraphthalate, polytetrafluoroethylene, latex andderivatives thereof, an elastomer, polyethylene tetraphthalate sealedwith gelatin, collagen or albumin, a cyanoacrylate, a methacrylate, apaper with porous barrier films, an adhesive, a linear copolymers, across-linked copolymer, a graft polymer, a block polymer, or acombination thereof.
 5. The composition according to claim 4, whereinsaid silicone is silicone elastomers, silicone adhesives or acombination thereof.
 6. The composition according to claim 4, whereinsaid polycarboxylic acid is selected from a group consisting ofpolyacrylic acid, polymethacrylic acid, polymaleic acid, poly-(maleicacid monoester), polyaspartic acid, polyglutamic acid, aginic acid,pectimic acid, or a combination thereof.
 7. The composition according toclaim 4, wherein said polycarboxylic acid anhydride is selected from agroup consisting of polymaleic anhydride, polymethacrylic anhydride,polyacrylic acid anhydride, or a combination thereof.
 8. The compositionaccording to claim 4, wherein said polyamine ions is selected from agroup consisting of polyethylene imine, polyvinylamine, polylysine,poly-(dialkylamineoethyl methacrylate), poly-(dialkylaminomethylstyrene), poly-(vinylpyridine), or a combination thereof.
 9. Thecomposition according to claim 4, wherein said polyammonium ion isselected from a group consisting of poly-(2-methacryloxyethyl trialkylammonium ion), poly-(vinylbenzyl trialkyl ammonium ions),poly-(N.N.-alkylypyridinium ion), poly-(dialkyloctamethylene ammoniumion), or a combination thereof.
 10. The composition according to claim4, wherein said polysulfonates is poly-(vinyl sulfonate), poly-(styrenesulfonate), or a combination thereof.
 11. The composition according toclaim 4, wherein said adhesive is selected from a group consisting ofhot melt adhesives, solvent based adhesives, adhesive hydrogels,fabrics, crosslinked hydrogels, non-crosslinked hydrogels, or acombination thereof.